The United States government has rights in this invention by virtue of National Institutes of Health grant No. CA42854.
This invention is generally in the area of methods for modifying nucleic acids, and more specifically relates to conversion of guanine to 8-hydroxyguanine using methylene blue.
Hydroxylation of guanine in DNA to produce 8-hydroxydeoxyguanosine (8-OH-dG) may be an important factor in mutation and carcinogenesis, as reportd and described by Kasai and Nishimura, Nucleic Acids Res. 12, 2137-2145 (1984); Gann. 75, 565-566and 841-844 (1984); Environ. Health Perspect. 67, 111-116 (1986); Kasai, et al., Gann. 75, 1037-1039 (1984); Carcinogenesis 7, 1849-1851 (1986); Aida and Nishimura, Mutation Res. 192, 83-89 (1987). Kuchino, et al., Nature (London) 327,77-79 (1987) used synthetic oligonucleotides containing 8-hydroxydeoxyguanosine in a specific position as a template for DNA synthesis to show misreading at both the modified base and at adjacent pyrimidine bases. They observed that specific base-pairing was completely lacking at the 8-hydroxyguanosine and that incorrect bases were inserted at the adjacent pyrimidine bases. Kasai, et al., reported in Carcinogenesis 8(12), 1959-1961 (1987) that administration of a renal carcinogen, potassium bromate, to the rat was followed by a significant increase of 8-hydroxydeoxyguanosine in the kidney DNA, and not in non-target organ DNA.
Chemically, 8-hydroxydeoxyguanosine is generated from guanosine by the action of reagents which generate oxygen radicals such as ascorbic acid and other reducing agents, metals, polyphenols, and asbestos and by x-irradiation. Ikehara, et al., reports in Chem. Parm. Bull. 13(9), 1140-1142 (1965) a method wherein guanosine is heated in acetic acid with an excess of sodium acetate to synthesize 8-hydroxyguanosine.
Intracellular DNA appears to undergo repair by enzymes following formation of 8-hydroxydeoxyguanosine. This may be a naturally occurring response which has evolved to combat the effects of the many mutagens, tumor promoters, and carcinogens which cause the formation of 8-hydroxydeoxyguanosine.
Methylene blue, 3,7-Bis(dimethylamino)phenothiazin-5-ium chloride, C.sub.16 H.sub.18 ClN.sub.3 S, is a dark green or blue thiazin dye which was first isolated in 1876. It is FDA approved for oral administration and has been reported to be effective as an antiseptic, disinfectant, and antidote for cyanide and nitrate poisoning. For over 50 years it has been known that methylene blue is reduced by mitochondria to leukodye which is then autooxidized back to methylene blue by oxygen, yielding H.sub.2 O.sub.2. This is the probable mechanism by which methylene blue, injected i.v. at a dose of 1 mg/kg body weight, is effective in the treatment of methemoglobinemia, a clinical disorder where more than 1% of the hemoglobin in the blood has been oxidized to Fe.sup.3 +. Kelner and Alexander reported in J. Biol. Chem. 260(28), 15168-15171 (1985), that methylene blue oxidizes glutathione directly when it is reduced by NADPH, rather than via the H.sub.2 O.sub.2.
Methylene blue, in the presence of light, has been reported to damage DNA, probably by damaging or cleaving the DNA at the guanine residues. In an effort to determine the mechanism by which certain photoactive dyes react with guanosine in the presence of light, Simon and Van Vunakis, Arch. Biochem. Biophys. 105, 197-206 (1964), noted that the effect of several photoactive dyes, including methylene blue, and light is dependent on the concentration of the dye, as well as light wavelength and intensity, and can be correlated with uptake of oxygen and decrease in ultraviolet absorbance by guanine derivatives.
Kornhauser, et al., Photochem. Photobiol. 18, 63-69 (1973) attempted to characterize the changes in guanosine following exposure to methylene blue and light using thin layer chromatographic analytical techniques.
Waskell, et al., reported in Biochim. Biophys. Acta 129, 49-53 (1966), that extensive irradiation of polynucleotides in the presence of methylene blue causes extensive destruction of the guanosine, leaving ribose, guanidine, ribosylurea, and free urea. They postulated that the destruction of the guanosine residues was the mechanism for a previous observation by Sastry, et al., Biochim. Biophys. Acta 129, 42 (1966), that methylene blue and irradiation inactivate TMV-RNA. Singer and Fraenkel-Conrat, had also reported, in Biochem. 4, 2446-2450 (1966), that another methylene-blue type dye, thiopyronin (where the ring N is replaced by CH), and proflavin cause inactivation of TMV RNA in the presence of light.
Others have attempted to analyze the effect of methylene blue and light on DNA, but without success. Friedmann and Brown, Nucleic Acids Res. 5, 615-622 (1978), showed that methylene blue and light caused lesions at deoxyguanosines in DNA and that subsequent exposure to piperidine caused strand rupture. They hypothesized that cyclo-addition occurred at various positions in the purine ring, rendering the DNA susceptible to base catalysed cleavage following modification of the other nucleoside bases.
It is therefore an object of the present invention to provide a method for using methylene blue and other thiazin dyes to selectively derivatize guanosine in a controlled manner.
It is a further object of the present invention to provide a method for mutating and cleaving both DNA and RNA in a selective manner using methylene blue and other thiazin dyes.
It is another object of the present invention to provide a method for selectively inactivating viruses and cancerous cells in vivo using methylene blue and other thiazin dyes.
It is a still further object of the present invention to provide methods and compositions for the selective delivery and utilization of methylene blue and other thiazin dyes in vivo.